Cellular environment is a major factor in determining cell behavior and ultimately cell phenotype. Many recent studies have shown that the mechanical aspects of the environment are important factors in determining the cell response. The mechanical factors that appear critical are the nanometer level spacing, curvature, and rigidity of the extracellular matrix components. We have formed a collaboration between two nanofabrication labs and a cell biology lab to explore how these factors are sensed by cells at the molecular level. Using an e-beam writing system, we will be able to create cellular sized arrays with a range of nanometer level features that will enable us to screen for the critical distances and patterns that trigger cell responses. Once we define the critical distance for a cell response such as cell spreading, we will screen a number of cell lines that are missing critical motility and signaling proteins to determine if the cell spreading response is altered. Of particular interest is talin that has multiple integrin binding sites spaced over about 55 nm. In regard to membrane curvature, we have observed that cells respond to collagen and other fibers by extending lamellipodia with myosin II-B in them and myosin II-B-/- cells contract fibers at less than 30% efficiency. We will fabricate fibers and 2-D surface features with different radii of curvature and use the assembly of GFP-myosin II-B in lamellipodia as a criterion for defining the range of curvatures that elicit the response. Cells not only follow fibers but also follow surface features through contact guidance and we will explore the important parameters in determining cell polarization and tracking. Recent results indicate that specific mutations will dramatically affect the ability of cells to polarize and move, giving rise to grossly abnormal morphologies that will enable us to understand important cellular parameters involved in contact guidance. Rigidity sensing is critical for cell spreading and growth and we have defined a number of proteins that are needed to sense the difference between rigid and soft surfaces. The development a device for probing the effect of changes in surface rigidity on adjacent regions of the cell surface will enable us to probe the detailed mechanism of rigidity sensing. Knowing both the size and geometry of surface features that are sensed by the cells will enable us to elicit given cell behaviors in a defined way. Knowing the proteins involved will enable us to use targeted pharmacological inhibitors to alter morphology in defined ways. These studies have many practical applications in tissue engineering and in designing potential therapies for wound healing, cancer and a variety of disorders.